Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

The present invention relates to therapeutic targets for aging. In
particular, the present invention relates to the inhibition of the
kynurenine pathway of tryptophan metabolism to extend lifespan or provide
anti-aging benefits.

Claims:

1. A method of prolonging the lifespan of a subject or preventing or
reducing age-related symptoms, comprising: administering an agent that
inhibits the conversion of tryptophan into kynurenine and/or increases
elimination of kynurenine to a subject, wherein said administering
prolongs the lifespan of said subject relative to the lifespan in the
absence of said agent or wherein age-related symptoms are reduced or
prevented.

6. The method of claim 1, wherein said administering treats or prevents
one or more aging-associated medical or psychiatric disorders.

7. The method of claim 1, wherein said agent is selected from the group
consisting of a small molecule, a nucleic acid, and an antibody.

8. A method of identifying compounds that inhibit the conversion of
tryptophan into kynurenine and/or increases elimination of kynurenine,
comprising: a) contacting a cell with a test compound; and b) identifying
test compounds that inhibit the conversion of tryptophan into
kynurenineor increase elimination of kynurenine.

16. The method of claim 14, wherein said animal is a non-human mammal.

17. The method of claim 8, wherein said agent is selected from the group
consisting of a small molecule, a nucleic acid, and an antibody.

18. A composition, comprising: a) a first agent that inhibits the
conversion of tryptophan into kynurenine and/or increases elimination of
kynurenine; and b) a second agent known to be useful in the treatment of
one or more disorders of aging.

22. The composition of claim 21, wherein said agent is 5-methyl
tryptophan.

Description:

[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/550,652, filed on Oct. 24, 2011, which is herein
incorporated by reference in its entirety

FIELD OF THE INVENTION

[0002] The present invention relates to therapeutic targets for aging. In
particular, the present invention relates to the inhibition of the
kynurenine pathway of tryptophan metabolism to extend lifespan or provide
anti-aging benefits.

[0004] For example, over time, the heart muscle becomes less efficient,
working harder to pump the same amount of blood. In addition, blood
vessels lose some of their elasticity and hardened fatty deposits may
form on the inner walls of arteries (atherosclerosis). These changes make
arteries stiffer, causing the heart to work even harder to pump blood
through them. This can lead to high blood pressure (hypertension) and
other cardiovascular problems.

[0005] With age, bones tend to shrink in size and density, which weakens
them and makes them more susceptible to fracture. Muscles generally lose
strength and flexibility, and individuals may become less coordinated or
have trouble balancing.

[0006] In addition, constipation is more common in older adults. Many
factors can contribute to constipation, including a low-fiber diet, not
drinking enough fluids and lack of exercise. Various medications,
including diuretics and iron supplements, may contribute to constipation.
Certain medical conditions, including diabetes and irritable bowel
syndrome, may increase the risk of constipation as well.

[0007] Loss of bladder control (urinary incontinence) is common with
aging. Health problems such as obesity, frequent constipation and chronic
cough may contribute to incontinence, as can menopause, for women, and an
enlarged prostate, for men.

[0008] Further, memory tends to becomes less efficient with age, as the
number of cells (neurons) in the brain decreases. It may take longer to
learn new things or remember familiar words or names.

[0009] With age, the eyes are less able to produce tears, the retinas
thin, and the lenses gradually become less clear. Focusing on objects
that are close up may become more difficult. People may become more
sensitive to glare and have trouble adapting to different levels of
light. Hearing may dim somewhat as well, in particular hearing high
frequencies or following a conversation in a crowded room.

[0010] With less saliva to wash away bacteria, teeth and gums become
slightly more vulnerable to decay and infection. Teeth also may darken
slightly and become more brittle and easier to break.

[0011] With age, skin thins and becomes less elastic and more fragile and
bruises more easily. Decreased production of natural oils may make skin
drier and more wrinkled. Age spots can occur, and small growths called
skin tags are more common.

[0012] Maintaining a healthy weight or losing weight if overweight is more
difficult as one ages. Muscle mass tends to decrease with age, which
leads to an increase in fat.

[0013] With age, sexual needs, patterns and performance may change.
Illness or medication may affect the ability to enjoy sex. For women,
vaginal dryness can make sex uncomfortable. For men, impotence may become
a concern. It may take longer to get an erection, and erections may not
be as firm as they used to be.

[0014] Additional disorders include as sarcopenia, diastolic dysfunction,
immune deficiencies, and mobility problems. Age-related mobility problems
are a very serious issue with far reaching health consequences.
Age-related mobility problems lead to an increase in falls,
hospitalizations, and future requirements for a caregiver, and increase
the risk for depression, osteoporosis, arthritis, congestive heart
failure, muscle pain, stroke, dementia and death.

[0015] While treatments exist for some symptoms of age related disorders,
no treatments that address multiple functions at a molecular level are
available.

SUMMARY OF THE INVENTION

[0016] The present invention relates to therapeutic targets for aging. In
particular, the present invention relates to the inhibition of the
kynurenine pathway of tryptophan metabolism to extend lifespan or provide
anti-aging benefits.

[0017] For example, in some embodiments, the present invention provides
compositions and methods of prolonging the lifespan of a subject or
providing anti-aging benefits, comprising: administering an agent that
inhibits the conversion of tryptophan into kynurenine to a subject (e.g.,
wherein the administering prolongs the lifespan of the subject relative
to the lifespan in the absence of the agent or where the one or more
anti-aging benefits are achieved). In some embodiments, the agent
inhibits TRY 2,3-dioxygenase 2 (TDO), indoleamine 2,3-dioxygenase (IDO)
or the ATP binding cassette (ABC) transporter. For example, in some
embodiments, the agent is a small molecule (e.g., alpha-methyl tryptophan
or 5-methyl tryptophan), a nucleic acid (e.g., siRNA or antisense nucleic
acid), an antibody, etc. In some embodiments, the administering treats,
prevents reduces or retards one or more aging-associated medical or
psychiatric disorders or conditions.

[0018] In some embodiments, the present invention provides a composition
comprising an agent that inhibits the conversion of tryptophan into
kynurenine and/or increases elimination of kynurenine or its neurotoxic
metabolites (e.g., 3HK); and an agent known to be useful in treating one
or more disorders associated with aging. In some embodiments, the agents
are formulated in a single pharmaceutical composition.

[0019] Further embodiments of the present invention provide a method of
identifying compounds that inhibit the conversion of tryptophan into
kynurenine and/or increases elimination of kynurenine or its neurotoxic
metabolites (e.g., 3HK) (e.g., to prolong lifespan or treat or prevent
one or more aging-associated medical or psychiatric disorders),
comprising: contacting a cell with a test compound; and identifying test
compounds that inhibit the conversion of tryptophan into kynurenine
and/or increases elimination of kynurenine or its neurotoxic metabolites
(e.g., 3HK). In some embodiments, the agent inhibits TRY 2,3-dioxygenase
2 (TDO), indoleamine 2,3-dioxygenase (IDO) or the ATP binding cassette
(ABC) transporter. In some embodiments, the cell is a mammalian cell
(e.g., a human cell) or a drosophila cell. In some embodiments, the cell
is in an animal (e.g., drosophila or a non-human mammal). In some
embodiments, the agent is a small molecule, a nucleic acid (e.g., siRNA
or antisense nucleic acid), an antibody, etc. In some embodiments, two or
more agents working together (e.g., additively or synergistically) are
evaluated. In some embodiments, aging-associated medical disorders (e.g.,
aging-associated medical and psychiatric disorders) include, but are not
limited to, reduced cardiac output, atherosclerosis, high blood pressure,
mood & cognitive changes, memory loss, vision problems, hearing loss,
muscle wasting, decreased energy, abdominal fat/truncal obesity, weak
bones, problems with coordination and balance, digestive problems (e.g.,
constipation), urinary incontinence, diabetes, thin skin and skin
wrinkles, poor sleep, age related mobility problems and lessened sexual
performance.

[0022] To facilitate an understanding of the present invention, a number
of terms and phrases are defined below:

[0023] As used herein, the term "prolonging the lifespan of a subject"
refers to increasing the lifespan of a subject relative to the lifespan
in the absence of the agent (e.g., relative to the projected lifespan of
an individual, a population, an individual with certain diseases or
lifestyle choices, etc.).

[0024] As used herein, the term "aging-associated medical and psychiatric
disorders" refers to medical or psychiatric disorder typically associated
with or worsened by aging. Examples include, but are not limited to,
those disclosed herein.

[0025] As used herein, the term "inhibits the conversion of tryptophan
into kynurenine" refers to any method of inhibiting the conversion of
tryptophan into kynurenine. In some embodiments, the enzyme catalyzing
tryptophan conversion into kynurenine (e.g., TRY 2,3-dioxygenase 2 (TDO)
and/or indoleamine 2,3-dioxygenase (IDO)) is inhibited. In other
embodiments, tryptophan tramsmembrane transport that delivers tryptophan
inside the cell producing kynurenine (e.g., ATP binding cassette (ABC)
transporter) is inhibited. TDO or ABC transporter may be inhibited using
any suitable agent (e.g., via directly contacting TDO or ABC transporter
protein, contacting TDO or ABC transporter mRNA or genomic DNA, causing
conformational changes of TDO or ABC transporter polypeptides, decreasing
TDO or ABC transporter protein levels, or interfering with TDO or ABC
transporter interactions with signaling partners, and affecting the
expression of TDO or ABC transporter target genes). Inhibitors also
include molecules that indirectly regulate TDO or ABC transporter
biological activity by intercepting upstream signaling molecules. In some
embodiments, the inhibitor is 5-methyl tryptophan or alpha-methyl
tryptophan.

[0026] As used herein, the term "subject" refers to any animal (e.g., a
mammal), including, but not limited to, humans, non-human primates,
rodents, and the like, which is to be the recipient of a particular
treatment. Typically, the terms "subject" and "patient" are used
interchangeably herein in reference to a human subject.

[0027] As used herein, the term "non-human animals" refers to all
non-human animals including, but are not limited to, vertebrates such as
rodents, non-human primates, ovines, bovines, ruminants, lagomorphs,
porcines, caprines, equines, canines, felines, ayes, etc.

[0029] The term "gene" refers to a nucleic acid (e.g., DNA) sequence that
comprises coding sequences necessary for the production of a polypeptide,
precursor, or RNA (e.g., rRNA, tRNA). The polypeptide can be encoded by a
full length coding sequence or by any portion of the coding sequence so
long as the desired activity or functional properties (e.g., enzymatic
activity, ligand binding, signal transduction, immunogenicity, etc.) of
the full-length or fragment is retained. The term also encompasses the
coding region of a structural gene and the sequences located adjacent to
the coding region on both the 5' and 3' ends for a distance of about 1 kb
or more on either end such that the gene corresponds to the length of the
full-length mRNA. Sequences located 5' of the coding region and present
on the mRNA are referred to as 5' non-translated sequences. Sequences
located 3' or downstream of the coding region and present on the mRNA are
referred to as 3' non-translated sequences. The term "gene" encompasses
both cDNA and genomic forms of a gene. A genomic form or clone of a gene
contains the coding region interrupted with non-coding sequences termed
"introns" or "intervening regions" or "intervening sequences." Introns
are segments of a gene that are transcribed into nuclear RNA (hnRNA);
introns may contain regulatory elements such as enhancers. Introns are
removed or "spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The mRNA
functions during translation to specify the sequence or order of amino
acids in a nascent polypeptide.

[0030] As used herein, the term "gene expression" refers to the process of
converting genetic information encoded in a gene into RNA (e.g., mRNA,
rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the
enzymatic action of an RNA polymerase), and for protein encoding genes,
into protein through "translation" of mRNA. Gene expression can be
regulated at many stages in the process. "Up-regulation" or "activation"
refers to regulation that increases the production of gene expression
products (i.e., RNA or protein), while "down-regulation" or "repression"
refers to regulation that decrease production. Molecules (e.g.,
transcription factors) that are involved in up-regulation or
down-regulation are often called "activators" and "repressors,"
respectively.

[0031] In addition to containing introns, genomic forms of a gene may also
include sequences located on both the 5' and 3' end of the sequences that
are present on the RNA transcript. These sequences are referred to as
"flanking" sequences or regions (these flanking sequences are located 5'
or 3' to the non-translated sequences present on the mRNA transcript).
The 5' flanking region may contain regulatory sequences such as promoters
and enhancers that control or influence the transcription of the gene.
The 3' flanking region may contain sequences that direct the termination
of transcription, post-transcriptional cleavage and polyadenylation.

[0032] As used herein, the term "oligonucleotide," refers to a short
length of single-stranded polynucleotide chain. Oligonucleotides are
typically less than 200 residues long (e.g., between 15 and 100),
however, as used herein, the term is also intended to encompass longer
polynucleotide chains. Oligonucleotides are often referred to by their
length. For example a 24 residue oligonucleotide is referred to as a
"24-mer". Oligonucleotides can form secondary and tertiary structures by
self-hybridizing or by hybridizing to other polynucleotides. Such
structures can include, but are not limited to, duplexes, hairpins,
cruciforms, bends, and triplexes.

[0033] As used herein, the terms "complementary" or "complementarity" are
used in reference to polynucleotides (i.e., a sequence of nucleotides)
related by the base-pairing rules. For example, for the sequence "A-G-T,"
is complementary to the sequence "T-C-A." Complementarity may be
"partial," in which only some of the nucleic acids' bases are matched
according to the base pairing rules. Or, there may be "complete" or
"total" complementarity between the nucleic acids. The degree of
complementarity between nucleic acid strands has significant effects on
the efficiency and strength of hybridization between nucleic acid
strands. This is of particular importance in amplification reactions, as
well as detection methods that depend upon binding between nucleic acids.

[0034] As used herein the term "portion" when in reference to a nucleotide
sequence (as in "a portion of a given nucleotide sequence") refers to
fragments of that sequence. The fragments may range in size from four
nucleotides to the entire nucleotide sequence minus one nucleotide (10
nucleotides, 20, 30, 40, 50, 100, 200, etc.).

[0035] As used herein, the term "cell culture" refers to any in vitro
culture of cells. Included within this term are continuous cell lines
(e.g., with an immortal phenotype), primary cell cultures, transformed
cell lines, finite cell lines (e.g., non-transformed cells), and any
other cell population maintained in vitro.

[0036] As used, the term "eukaryote" refers to organisms distinguishable
from "prokaryotes." It is intended that the term encompass all organisms
with cells that exhibit the usual characteristics of eukaryotes, such as
the presence of a true nucleus bounded by a nuclear membrane, within
which lie the chromosomes, the presence of membrane-bound organelles, and
other characteristics commonly observed in eukaryotic organisms. Thus,
the term includes, but is not limited to such organisms as fungi,
protozoa, and animals (e.g., humans).

[0037] As used herein, the term "in vitro" refers to an artificial
environment and to processes or reactions that occur within an artificial
environment. In vitro environments can consist of, but are not limited
to, test tubes and cell culture. The term "in vivo" refers to the natural
environment (e.g., an animal or a cell) and to processes or reaction that
occur within a natural environment.

[0038] The terms "test compound" and "candidate compound" refer to any
chemical entity, pharmaceutical, drug, and the like that is a candidate
for use to treat or prevent a disease, illness, sickness, or disorder of
bodily function (e.g., medical or psychiatric disorders of aging). Test
compounds comprise both known and potential therapeutic compounds. A test
compound can be determined to be therapeutic by screening using the
screening methods of the present invention. In some embodiments of the
present invention, test compounds include antisense compounds.

[0039] As used herein, the term "sample" is used in its broadest sense. In
one sense, it is meant to include a specimen or culture obtained from any
source, as well as biological and environmental samples. Biological
samples may be obtained from animals (including humans) and encompass
fluids, solids, tissues, and gases. Biological samples include blood
products, such as plasma, serum and the like. Environmental samples
include environmental material such as surface matter, soil, water, and
industrial samples. Such examples are not however to be construed as
limiting the sample types applicable to the present invention.

[0041] The term "derivative" of a compound, as used herein, refers to a
chemically modified compound wherein the chemical modification takes
place either at a functional group of the compound or backbone. Such
derivatives include, but are not limited to, esters of alcohol-containing
compounds, esters of carboxy-containing compounds, amides of
amine-containing compounds, amides of carboxy-containing compounds,
imines of amino-containing compounds, acetals of aldehyde-containing
compounds, ketals of carbonyl-containing compounds, and the like.

[0042] As used herein, the term "pharmaceutically acceptable salt" refers
to any pharmaceutically acceptable salt (e.g., acid or base) of a
compound of the present invention which, upon administration to a
subject, is capable of providing a compound of this invention or an
active metabolite or residue thereof. As is known to those of skill in
the art, "salts" of the compounds of the present invention may be derived
from inorganic or organic acids and bases. Examples of acids include, but
are not limited to, hydrochloric, hydrobromic, sulfuric, nitric,
perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic,
succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,
ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,
benzenesulfonic acid, and the like. Other acids, such as oxalic, while
not in themselves pharmaceutically acceptable, may be employed in the
preparation of salts useful as intermediates in obtaining the compounds
of the invention and their pharmaceutically acceptable acid addition
salts.

[0045] For therapeutic use, salts of the compounds of the present
invention are contemplated as being pharmaceutically acceptable. However,
salts of acids and bases that are non-pharmaceutically acceptable may
also find use, for example, in the preparation or purification of a
pharmaceutically acceptable compound.

[0046] As used herein, the term "siRNAs" refers to small interfering RNAs.
In some embodiments, siRNAs comprise a duplex, or double-stranded region,
of about 18-25 nucleotides long; often siRNAs contain from about two to
four unpaired nucleotides at the 3' end of each strand. At least one
strand of the duplex or double-stranded region of a siRNA is
substantially homologous to, or substantially complementary to, a target
RNA molecule. The strand complementary to a target RNA molecule is the
"antisense strand;" the strand homologous to the target RNA molecule is
the "sense strand," and is also complementary to the siRNA antisense
strand. siRNAs may also contain additional sequences; non-limiting
examples of such sequences include linking sequences, or loops, as well
as stem and other folded structures. siRNAs appear to function as key
intermediaries in triggering RNA interference in invertebrates and in
vertebrates, and in triggering sequence-specific RNA degradation during
posttranscriptional gene silencing in plants.

[0047] The term "RNA interference" or "RNAi" refers to the silencing or
decreasing of gene expression by siRNAs. It is the process of
sequence-specific, post-transcriptional gene silencing in animals and
plants, initiated by siRNA that is homologous in its duplex region to the
sequence of the silenced gene. The gene may be endogenous or exogenous to
the organism, present integrated into a chromosome or present in a
transfection vector that is not integrated into the genome. The
expression of the gene is either completely or partially inhibited. RNAi
may also be considered to inhibit the function of a target RNA; the
function of the target RNA may be complete or partial.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention relates to therapeutic targets for aging. In
particular, the present invention relates to the inhibition of the
kynurenine pathway of tryptophan metabolism to extend lifespan or provide
anti-aging benefits.

[0049] Embodiments of the present invention provide compositions and
methods of prolonging lifespan in a subject or otherwise providing
anti-aging benefits by inhibiting the conversion of tryptophan (TRY) into
kynurenine (KYN) and/or increases elimination of kynurenine or its
neurotoxic metabolites (e.g., 3HK). For example, in some embodiments the
enzyme catalyzing tryptophan conversion into kynurenine (e.g., TRY
2,3-dioxygenase 2 (TDO)) is inhibited. In other embodiments, tryptophan
tramsmembrane transport that delivers tryptophan inside the cell
producing kynurenine (e.g., ATP binding cassette (ABC) transporter) is
inhibited.

[0052] TRY-KYN pathway and related genes were described in Drosophila
melanogaster (Savvateeva-Popova et al. 2003 Adv. Exp. Med. Biol. 527,
713-722). The end product of TRY-KYN pathway in Drosophila is brown eye
pigment (Tearle, 1991 Genet. Res. 57, 257-266). TDO is the rate-limiting
enzyme of KYN formation from TRY in Drosophila, as in the other species.
Experiments conducted during the course of development of embodiments of
the present invention investigated whether prolongation of life span was
associated with the slow rate of KYN formation from TRY. In difference
with genetic mutations, pharmacological interventions increased not only
mean survival time but maximum life span as well.

I. Therapeutic Applications

[0053] As described above, embodiments of the present invention provide
compositions and methods of inhibiting TRY conversion to KYN and/or
increases elimination of kynurenine or its neurotoxic metabolites (e.g.,
3HK) (e.g., by inhibiting TRY 2,3-dioxygenase 2 (TDO), indoleamine
2,3-dioxygenase (IDO) or ATP binding cassette (ABC) transporter). A
number of exemplary methods of inhibiting TDO and ABC transporter are
described herein. One of skill in the art recognizes that other
therapeutics are suitable for use herein.

A. Small Molecule Therapies

[0054] In other embodiments, the present invention provides small molecule
inhibitors of TDO, IDO and/or ABC transporter expression or activity.

[0055] In some embodiments, the small molecule therapeutic is the TDO
inhibitor, alpha-methyl tryptophan (aMT) or the ABC transporter
inhibitor, 5-methyl tryptophan (5MT).

[0056] In some embodiments, the inhibitor is a mimetic, derivative,
analog, stereoisomer, etc. of aMT or 5MT. Additional small molecule
therapeutics can be identified using the drug screening methods described
herein.

[0057] The present invention also includes pharmaceutical compositions and
formulations that include the small molecule compounds of the present
invention as described below.

B. RNA Interference and Antisense Therapies

[0058] In some embodiments, the present invention targets the expression
of TDO, IDO or ABC transporter. For example, in some embodiments, the
present invention employs compositions comprising oligomeric antisense or
RNAi compounds, particularly oligonucleotides (e.g., those described
herein), for use in modulating the function of nucleic acid molecules
encoding TDO, IDO or ABC transporter, ultimately modulating the amount of
TDO, IDO or ABC transporter expressed.

[0059] 1. RNA Interference (RNAi)

[0060] In some embodiments, RNAi is utilized to inhibit TDO, IDO or ABC
transporter protein function. RNAi represents an evolutionary conserved
cellular defense for controlling the expression of foreign genes in most
eukaryotes, including humans. RNAi is typically triggered by
double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation
of single-stranded target RNAs homologous in response to dsRNA. The
mediators of mRNA degradation are small interfering RNA duplexes
(siRNAs), which are normally produced from long dsRNA by enzymatic
cleavage in the cell. siRNAs are generally approximately twenty-one
nucleotides in length (e.g. 21-23 nucleotides in length), and have a
base-paired structure characterized by two nucleotide 3'-overhangs.
Following the introduction of a small RNA, or RNAi, into the cell, it is
believed the sequence is delivered to an enzyme complex called
RISC(RNA-induced silencing complex). RISC recognizes the target and
cleaves it with an endonuclease. It is noted that if larger RNA sequences
are delivered to a cell, RNase III enzyme (Dicer) converts longer dsRNA
into 21-23 nt ds siRNA fragments.

[0062] siRNAs are extraordinarily effective at lowering the amounts of
targeted RNA, and by extension proteins, frequently to undetectable
levels. The silencing effect can last several months, and is
extraordinarily specific, because one nucleotide mismatch between the
target RNA and the central region of the siRNA is frequently sufficient
to prevent silencing (Brummelkamp et al, Science 2002; 296:550-3; and
Holen et al, Nucleic Acids Res. 2002; 30:1757-66, both of which are
herein incorporated by reference).

[0063] An important factor in the design of siRNAs is the presence of
accessible sites for siRNA binding. Bahoia et al., (J. Biol. Chem., 2003;
278: 15991-15997; herein incorporated by reference) describe the use of a
type of DNA array called a scanning array to find accessible sites in
mRNAs for designing effective siRNAs. These arrays comprise
oligonucleotides ranging in size from monomers to a certain maximum,
usually Corners, synthesized using a physical barrier (mask) by stepwise
addition of each base in the sequence. Thus the arrays represent a full
oligonucleotide complement of a region of the target gene. Hybridization
of the target mRNA to these arrays provides an exhaustive accessibility
profile of this region of the target mRNA. Such data are useful in the
design of antisense oligonucleotides (ranging from 7mers to 25mers),
where it is important to achieve a compromise between oligonucleotide
length and binding affinity, to retain efficacy and target specificity
(Sohail et al, Nucleic Acids Res., 2001; 29(10): 2041-2045). Additional
methods and concerns for selecting siRNAs are described for example, in
WO 05054270, WO05038054A1, WO03070966A2, J Mol. Biol. 2005 May 13;
348(4):883-93, J Mol. Biol. 2005 May 13; 348(4):871-81, and Nucleic Acids
Res. 2003 Aug. 1; 31(15):4417-24, each of which is herein incorporated by
reference in its entirety. In addition, software (e.g., the MWG online
siMAX siRNA design tool) is commercially or publicly available for use in
the selection of siRNAs.

[0064] In some embodiments, the present invention utilizes siRNA including
blunt ends (See e.g., US20080200420, herein incorporated by reference in
its entirety), overhangs (See e.g., US20080269147A1, herein incorporated
by reference in its entirety), locked nucleic acids (See e.g.,
WO2008/006369, WO2008/043753, and WO2008/051306, each of which is herein
incorporated by reference in its entirety). In some embodiments, siRNAs
are delivered via gene expression or using bacteria (See e.g., Xiang et
al., Nature 24: 6 (2006) and WO06066048, each of which is herein
incorporated by reference in its entirety).

[0065] In other embodiments, shRNA techniques (See e.g., 20080025958,
herein incorporated by reference in its entirety) are utilized. A small
hairpin RNA or short hairpin RNA (shRNA) is a sequence of RNA that makes
a tight hairpin turn that can be used to silence gene expression via RNA
interference. shRNA uses a vector introduced into cells and utilizes the
U6 promoter to ensure that the shRNA is always expressed. This vector is
usually passed on to daughter cells, allowing the gene silencing to be
inherited. The shRNA hairpin structure is cleaved by the cellular
machinery into siRNA, which is then bound to the RNA-induced silencing
complex (RISC). This complex binds to and cleaves mRNAs which match the
siRNA that is bound to it. shRNA is transcribed by RNA polymerase III.

[0066] The present invention also includes pharmaceutical compositions and
formulations that include the RNAi compounds of the present invention as
described below.

[0067] 2. Antisense

[0068] In other embodiments, TDO, IDO or ABC transporter protein
expression is modulated using antisense compounds that specifically
hybridize with one or more nucleic acids encoding TDO, IDO or ABC
transporter. The specific hybridization of an oligomeric compound with
its target nucleic acid interferes with the normal function of the
nucleic acid. This modulation of function of a target nucleic acid by
compounds that specifically hybridize to it is generally referred to as
"antisense." The functions of DNA to be interfered with include
replication and transcription. The functions of RNA to be interfered with
include all vital functions such as, for example, translocation of the
RNA to the site of protein translation, translation of protein from the
RNA, splicing of the RNA to yield one or more mRNA species, and catalytic
activity that may be engaged in or facilitated by the RNA. The overall
effect of such interference with target nucleic acid function is
modulation of the expression of TDO, IDO or ABC transporter. In the
context of the present invention, "modulation" means either an increase
(stimulation) or a decrease (inhibition) in the expression of a gene. For
example, expression may be inhibited to prevent symptoms related to
disorders of aging and thus prolong lifespan.

[0069] The present invention also includes pharmaceutical compositions and
formulations that include the antisense compounds of the present
invention as described below.

C. Genetic Therapy

[0070] The present invention contemplates the use of any genetic
manipulation for use in modulating the expression of TDO, IDO or ABC
transporter. Examples of genetic manipulation include, but are not
limited to, gene knockout (e.g., removing the TDO, IDO or ABC transporter
gene from the chromosome using, for example, recombination), expression
of antisense constructs with or without inducible promoters, and the
like. Delivery of nucleic acid construct to cells in vitro or in vivo may
be conducted using any suitable method. A suitable method is one that
introduces the nucleic acid construct into the cell such that the desired
event occurs (e.g., expression of an antisense construct). Genetic
therapy may also be used to deliver siRNA or other interfering molecules
that are expressed in vivo (e.g., upon stimulation by an inducible
promoter (e.g., an androgen-responsive promoter)).

[0071] Introduction of molecules carrying genetic information into cells
is achieved by any of various methods including, but not limited to,
directed injection of naked DNA constructs, bombardment with gold
particles loaded with said constructs, and macromolecule mediated gene
transfer using, for example, liposomes, biopolymers, and the like.
Preferred methods use gene delivery vehicles derived from viruses,
including, but not limited to, adenoviruses, retroviruses, vaccinia
viruses, and adeno-associated viruses. Because of the higher efficiency
as compared to retroviruses, vectors derived from adenoviruses are the
preferred gene delivery vehicles for transferring nucleic acid molecules
into host cells in vivo. Adenoviral vectors have been shown to provide
very efficient in vivo gene transfer into a variety of tissues in animal
models. Examples of adenoviral vectors and methods for gene transfer are
described in PCT publications WO 00/12738 and WO 00/09675 and U.S. Pat.
Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128, 5,994,106,
5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of which
is herein incorporated by reference in its entirety.

[0072] Vectors may be administered to subjects in a variety of ways. For
example, in some embodiments of the present invention, vectors are
administered using direct injection. In other embodiments, administration
is via the blood or lymphatic circulation (See e.g., PCT publication
99/02685 herein incorporated by reference in its entirety). Exemplary
dose levels of adenoviral vector are preferably 108 to 1011
vector particles added to the perfusate.

D. Pharmaceutical Compositions

[0073] The compounds are preferably employed for therapeutic uses in
combination with a suitable pharmaceutical carrier. Such compositions
comprise an effective amount of the compound, and a pharmaceutically
acceptable carrier or excipient. The formulation is made to suit the mode
of administration. Pharmaceutically acceptable carriers are determined in
part by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly, there
is a wide variety of suitable formulations of pharmaceutical compositions
containing the nucleic acids some of which are described herein.

[0074] The compounds may be in a formulation for administration topically,
locally or systemically in a suitable pharmaceutical carrier. Remington's
Pharmaceutical Sciences, 15th Edition by E. W. Martin (Mark Publishing
Company, 1975), discloses typical carriers and methods of preparation.
The compound may also be encapsulated in suitable biocompatible
microcapsules, microparticles or micro spheres formed of biodegradable or
non-biodegradable polymers or proteins or liposomes for targeting to
cells. Such systems are well known to those skilled in the.

[0075] Formulations suitable for parenteral administration, such as, for
example, by intraarticular (in the joints), intravenous, intramuscular,
intradermal, intraperitoneal, and subcutaneous routes, include aqueous
and non-aqueous, isotonic sterile injection solutions, which can contain
antioxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and
aqueous and non-aqueous sterile suspensions, solutions or emulsions that
can include suspending agents, solubilizers, thickening agents,
dispersing agents, stabilizers, and preservatives. Formulations for
injection may be presented in unit dosage form, e.g., in ampules or in
multi-dose containers, with an added preservative.

[0076] Preparations include sterile aqueous or nonaqueous solutions,
suspensions and emulsions, which can be isotonic with the blood of the
subject in certain embodiments. Examples of nonaqueous solvents are
polypropylene glycol, polyethylene glycol, vegetable oil such as olive
oil, sesame oil, coconut oil, arachis oil, peanut oil, mineral oil,
injectable organic esters such as ethyl oleate, or fixed oils including
synthetic mono or di-glycerides. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including saline
and buffered media. Parenteral vehicles include sodium chloride solution,
1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also be
present such as, for example, antimicrobials, antioxidants, chelating
agents and inert gases and the like. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic mono- or
di-glycerides. In addition, fatty acids such as oleic acid may be used in
the preparation of injectables. Carrier formulation can be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
Those of skill in the art can readily determine the various parameters
for preparing and formulating the compositions without resort to undue
experimentation.

[0077] The compound alone or in combination with other suitable
components, can also be made into aerosol formulations (i.e., they can be
"nebulized") to be administered via inhalation. Aerosol formulations can
be placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. For
administration by inhalation, the compounds are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or a
nebulizer, with the use of a suitable propellant.

[0078] In some embodiments, the compound described above may include
pharmaceutically acceptable carriers with formulation ingredients such as
salts, carriers, buffering agents, emulsifiers, diluents, excipients,
chelating agents, fillers, drying agents, antioxidants, antimicrobials,
preservatives, binding agents, bulking agents, silicas, solubilizers, or
stabilizers. In one embodiment, the compounds are conjugated to
lipophilic groups like cholesterol and laurie and lithocholic acid
derivatives with C32 functionality to improve cellular uptake. For
example, cholesterol has been demonstrated to enhance uptake and serum
stability of siRNA in vitro Lorenz, et al., Bioorg. Med. Chem. Lett.
14(19):4975-4977 (2004)) and in vivo (Soutschek, et al., Nature
432(7014):173-178 (2004)). In addition, it has been shown that binding of
steroid conjugated oligonucleotides to different lipoproteins in the
bloodstream, such as LDL, protect integrity and facilitate
biodistribution (Rump, et al., Biochem. Pharmacol. 59 (11):1407-1416
(2000)). Other groups that can be attached or conjugated to the compound
described above to increase cellular uptake, include acridine
derivatives; cross-linkers such as psoralen derivatives, azidophenacyl,
proflavin, and azidoproflavin; artificial endonucleases; metal complexes
such as EDTA-Fe(II) and porphyrin-Fe(II); alkylating moieties; nucleases
such as alkaline phosphatase; terminal transferases; abzymes; cholesteryl
moieties; lipophilic carriers; peptide conjugates; long chain alcohols;
phosphate esters; radioactive markers; non-radioactive markers;
carbohydrates; and polylysine or other polyamines.

[0079] U.S. Pat. No. 6,919,208 to Levy, et al., herein incorporated by
reference, also described methods for enhanced delivery. These
pharmaceutical formulations may be manufactured in a manner that is
itself known, e.g., by means of conventional mixing, dissolving,
granulating, levigating, emulsifying, encapsulating, entrapping or
lyophilizing processes.

[0080] Compositions can be administered by a number of routes including,
but not limited to: oral, intravenous, intraperitoneal, intramuscular,
transdermal, subcutaneous, topical, sublingual, or rectal means.
Compounds can also be administered via liposomes. Such administration
routes and appropriate formulations are generally known to those of skill
in the art.

[0081] The particular mode selected will depend of course, upon factors
such as the particular formulation, the severity of the state of the
subject being treated, and the dosage required for therapeutic efficacy.
As generally used herein, an "effective amount" is that amount which is
able to treat one or more symptoms of aging related disorders, reverse
the progression of one or more symptoms of aging related disorders, halt
the progression of one or more symptoms of aging related disorders, or
prevent the occurrence of one or more symptoms of aging related disorders
in a subject to whom the formulation is administered, as compared to a
matched subject not receiving the compound.

[0082] The actual effective amounts of compound can vary according to the
specific compound or combination thereof being utilized, the particular
composition formulated, the mode of administration, and the age, weight,
condition of the individual, and severity of the symptoms or condition
being treated.

[0083] Any acceptable method known to one of ordinary skill in the art may
be used to administer a formulation to the subject. The administration
may be localized (i.e., to a particular region, physiological system,
tissue, organ, or cell type) or systemic, depending on the condition
being treated.

[0084] Injections can be e.g., intravenous, intradermal, subcutaneous,
intramuscular, or intraperitoneal. The composition can be injected
intraderinally for treatment or prevention of aging related disorders,
for example. In some embodiments, the injections can be given at multiple
locations. Implantation includes inserting implantable drug delivery
systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol
matrixes, polymeric systems, e.g., matrix erosion and/or diffusion
systems and non-polymeric systems, e.g., compressed, fused, or
partially-fused pellets. Inhalation includes administering the
composition with an aerosol in an inhaler, either alone or attached to a
carrier that can be absorbed. For systemic administration, it may be
preferred that the composition is encapsulated in liposomes.

[0085] Other delivery systems suitable include time-release, delayed
release, sustained release, or controlled release delivery systems. Such
systems may avoid repeated administrations in many cases, increasing
convenience to the subject and the physician. Many types of release
delivery systems are available and known to those of ordinary skill in
the art. They include, for example, polymer-based systems such as
polylactic and/or polyglycolic acids, polyanhydrides, polycaprolactones,
copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric
acid, and/or combinations of these.

[0086] Use of a long-term release implant may be particularly suitable in
some embodiments. "Long-term release," as used herein, means that the
implant containing the composition is constructed and arranged to deliver
therapeutically effective levels of the composition for at least 30 or 45
days, and preferably at least 60 or 90 days, or even longer in some
cases. Long-term release implants are well known to those of ordinary
skill in the art, and include some of the release systems described
above.

[0087] Dosages for a particular individual can be determined by one of
ordinary skill in the art using conventional considerations, (e.g. by
means of an appropriate, conventional pharmacological protocol). A
physician may, for example, prescribe a relatively low dose at first,
subsequently increasing the dose until an appropriate response is
obtained. The dose administered to a individual is sufficient to effect a
beneficial therapeutic response in the individual over time, or, e.g., to
reduce symptoms, or other appropriate activity, depending on the
application. The dose is determined by the efficacy of the particular
formulation, and the activity, stability or serum half-life of the
therapeutic employed and the condition of the individual, as well as the
body weight or surface area of the individual to be treated. The size of
the dose is also determined by the existence, nature, and extent of any
adverse side-effects that accompany the administration of a particular
vector, formulation, or the like in a particular individual.

E. Co-Administration

[0088] In some embodiments, the present invention provides combination
therapies. The formulations described herein can supplement treatment
conditions by any known conventional therapy, including, but not limited
to, antibody administration, vaccine administration, administration of
cytotoxic agents, natural amino acid polypeptides, nucleic acids,
nucleotide analogues, and biologic response modifiers. Two or more
combined compounds may be used together or sequentially. For example,
agents can also be administered in therapeutically effective amounts as a
portion of an anti-age-related disorder cocktail.

[0089] For example, in some embodiments, one or more of the therapeutic
compositions described herein are combined with a known anti-aging agent.
In some embodiments, compounds for co-administration are formulated
together in a composition (e.g., pill, tablet, liquid, injectible
formulation, etc). In other embodiments, compounds are separately
formulated but administered to the same subject.

II. Antibodies

[0090] The present invention provides isolated antibodies. In some
embodiments, the present invention provides monoclonal antibodies that
specifically bind to an isolated polypeptide comprised of at least five
amino acid residues of TDO, IDO or ABC transporter. These antibodies find
use in the therapeutic and drug screening methods described herein.

[0091] An antibody against a protein of the present invention may be any
monoclonal or polyclonal antibody, as long as it can recognize the
protein. Antibodies can be produced by using a protein of the present
invention as the antigen according to a conventional antibody or
antiserum preparation process.

[0092] The present invention contemplates the use of both monoclonal and
polyclonal antibodies. Any suitable method may be used to generate the
antibodies used in the methods and compositions of the present invention,
including but not limited to, those disclosed herein. For example, for
preparation of a monoclonal antibody, protein, as such, or together with
a suitable carrier or diluent is administered to an animal (e.g., a
mammal) under conditions that permit the production of antibodies. For
enhancing the antibody production capability, complete or incomplete
Freund's adjuvant may be administered. Normally, the protein is
administered once every 2 weeks to 6 weeks, in total, about 2 times to
about 10 times. Animals suitable for use in such methods include, but are
not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep,
goats, etc.

III. Drug Screening Applications

[0093] In some embodiments, the present invention provides drug screening
assays (e.g., to screen for drugs that inhibit TDO, IDO or ABC
transporter). In some embodiments, the screening methods of the present
invention utilize TDO, IDO or ABC transporter or a measure of their
activity or expression. For example, in some embodiments, the present
invention provides methods of screening for compounds that alter (e.g.,
decrease) the expression or activity of TDO, IDO or ABC transporter. The
compounds or agents may interfere with transcription, by interacting, for
example, with the promoter region. The compounds or agents may interfere
with mRNA produced from TDO, IDO or ABC transporter (e.g., by RNA
interference, antisense technologies, etc.). The compounds or agents may
interfere with pathways that are upstream or downstream of the biological
activity of TDO, IDO or ABC transporter. In some embodiments, candidate
compounds are antisense or interfering RNA agents (e.g.,
oligonucleotides) directed against TDO or ABC transporter. In other
embodiments, candidate compounds are antibodies or small molecules that
specifically bind to an TDO, IDO or ABC transporter regulator or
expression products of the present invention and inhibit its biological
function.

[0094] In one screening method, candidate compounds are evaluated for
their ability to alter TDO, IDO or ABC transporter expression by
contacting a compound with a cell expressing TDO, IDO or ABC transporter
and then assaying for the effect of the candidate compounds on
expression. In some embodiments, the effect of candidate compounds on
expression of an TDO, IDO or ABC transporter gene is assayed for by
detecting the level of TDO, IDO or ABC transporter mRNA expressed by the
cell. mRNA expression can be detected by any suitable method.

[0095] In other embodiments, the effect of candidate compounds on
expression of TDO, IDO or ABC transporter genes is assayed by measuring
the level of polypeptide encoded by TDO, IDO or ABC transporter. The
level of polypeptide expressed can be measured using any suitable method,
including but not limited to, those disclosed herein.

[0096] Specifically, the present invention provides screening methods for
identifying modulators, i.e., candidate or test compounds or agents
(e.g., proteins, peptides, peptidomimetics, peptoids, small molecules or
other drugs) which bind to TDO, IDO or ABC transporter, have an
inhibitory (or stimulatory) effect on, for example, TDO, IDO or ABC
transporter expression or TDO, IDO or ABC transporter activity, or have a
stimulatory or inhibitory effect on, for example, the expression or
activity of a TDO, IDO or ABC transporter substrate. Compounds thus
identified can be used to modulate the activity of target gene products
(e.g., TDO, IDO or ABC transporter) either directly or indirectly in a
therapeutic protocol, to elaborate the biological function of the target
gene product, or to identify compounds that disrupt normal target gene
interactions. Compounds that inhibit the activity or expression of TDO,
IDO or ABC transporter are useful in the treatment of aging related
disorders.

[0097] In one embodiment, the invention provides assays for screening
candidate or test compounds that are substrates of an TDO, IDO or ABC
transporter protein or polypeptide or a biologically active portion
thereof. In another embodiment, the invention provides assays for
screening candidate or test compounds that bind to or modulate the
activity of an TDO or ABC transporter protein or polypeptide or a
biologically active portion thereof.

[0098] The test compounds of the present invention can be obtained using
any of the numerous approaches in combinatorial library methods known in
the art, including biological libraries; peptoid libraries (libraries of
molecules having the functionalities of peptides, but with a novel,
non-peptide backbone, which are resistant to enzymatic degradation but
which nevertheless remain bioactive; see, e.g., Zuckennann et al., J.
Med. Chem. 37: 2678-85

[0101] The following examples are provided in order to demonstrate and
further illustrate certain preferred embodiments and aspects of the
present invention and are not to be construed as limiting the scope
thereof.

Example 1

Methods

[0102] Wild type Oregon-R flies were maintained at 23° C. on a
standard Drosophila medium consisting of sugar, yeast, agar and semolina.
Two concentrations of aMT (alpha-dl-methyl tryptophan) (0.46 mM or 18.3
mM) or 5MT (5-methyl-dl-tryptophan) (2.4 mM and 18.mM) (Sigma Aldrich
Chemical Co, USA) were added to nutrition medium of experimental groups.
To examine lifespan, 1-day old adult flies were collected and then
regularly transferred to fresh medium every 3-4 days. The number of dead
flies was recorded at the time of transfer.

Statistics

[0103] The data were analyzed using two ways ANOVA and Log rank test.

Results

Effect of Alpha-Methyl Tryptophan

[0104] Treatment with aMT (0.46 mM) did not affect the life span of
Drosophila. Treatment with higher concentration of aMT (18.3 mM)
increased mean survival time by 27% for females and by 42% for males
(p<0.0001, two way ANOVA) (Table 1). Treatment with high concentration
of aMT (18.3 mM) increased maximum life span in both female and male
flies by 23% and 21% (resp.)(p<0.0001, two way ANOVA) (Table 1).
Maximum life span of female control group was 53 days. 25% (13 out of 51)
female flies treated with high concentrations of aMT survived longer than
53 days (up to 65 days) (FIG. 1). Maximum life span of 72 out of 73 male
control flies was 40 days. Only one control fly (1.3%) lived up to 46
days while 60% (29 out of 40) male flies treated with high concentrations
of aMT survived longer than 40 days (up to 56 days) (FIG. 1).

Effect of 5-Methyl Tryptophan.

[0105] Treatment with 5MT (2.3 mM) did not affect the life span of
Drosophila. Treatment with high concentration of aMT (18.3 mM) did not
affect life span of male flies (Table 1). Treatment with higher
concentration of 5MT (34.5 mM) increased mean survival time (by 21%) and
maximum life span (by 23%) of female flies (p<0.0001, two way ANOVA)
(Table 1). Maximum life span of female control group was 53 days. 19% (11
out of 58) female flies treated with high concentrations of 5MT survived
longer than 53 days (up to 65 days).

[0106] All publications and patents mentioned in the above specification
are herein incorporated by reference. Various modifications and
variations of the described method and system of the invention will be
apparent to those skilled in the art without departing from the scope and
spirit of the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be understood
that the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the described
modes for carrying out the invention which are obvious to those skilled
in the relevant fields are intended to be within the scope of the
following claims.

Patent applications by Gregory Oxenkrug, Newton, MA US

Patent applications in class IMMUNOGLOBULIN, ANTISERUM, ANTIBODY, OR ANTIBODY FRAGMENT, EXCEPT CONJUGATE OR COMPLEX OF THE SAME WITH NONIMMUNOGLOBULIN MATERIAL

Patent applications in all subclasses IMMUNOGLOBULIN, ANTISERUM, ANTIBODY, OR ANTIBODY FRAGMENT, EXCEPT CONJUGATE OR COMPLEX OF THE SAME WITH NONIMMUNOGLOBULIN MATERIAL